Comprehensive Characterization of Tense and Relaxed State Glutaraldehyde
Polymerized Bovine Hemoglobin as a Function of Cross-link Density
Abstract
Previously, our lab developed high molecular weight (MW) tense (T) state
glutaraldehyde polymerized bovine hemoglobins (PolybHbs) that exhibited
reduced vasoactivity in several small animal models. In this work, we
prepared PolybHb in the T- and relaxed (R) quaternary state with
ultrahigh MW (> 500 kDa) with varying cross-link densities
and investigated the effect of MW on key biophysical properties (i.e.,
O2 affinity, cooperativity coefficient, hydrodynamic diameter,
polydispersity, polymer composition, viscosity, gaseous ligand-binding
kinetics, autoxidation, and haptoglobin-binding kinetics). To further
optimize current PolybHb synthesis and purification protocols, we
performed a comprehensive meta-data analysis to evaluate correlations
between procedural parameters (i.e. cross-linker:bovine Hb (bHb) molar
ratio, gas/liquid exchange time, temperature during dithionite addition,
and number of diafiltration cycles) and the biophysical properties of
both T-state and R-state PolybHbs. Our results showed that, the duration
of the fast-step autoxidation phase of R-state PolybHb increased with
decreasing glutaraldehyde:bHb molar ratio. Additionally, T-state PolybHb
exhibited significantly higher biomolecular rate constants for binding
to haptoglobin and unimoleular O2 offloading rate constants compared to
R-state PolybHb. The methemoglobin (metHb) level in the final product
was insensitive to the molar ratio of glutaraldehyde to bHb for all
PolybHb. During tangential flow filtration processing of the final
product, 14 diafiltration cycles was found to yield the lowest metHb
level.